This invention describes an improved method to conduct proximity measurements based on the detection of transfer of excitation energy between species labelled with luminescent chromophores.
Luminescence techniques (exemplified by, but riot limited to, fluorescence and phosphorescence) are widely used in a variety of analytical applications. There is much interest in using luminescent labels as replacements for radioisotopes in assays and measurements where very high sensitivity is required. Luminescent labels are also used widely in a number of measurements where proximity between labelled species is to be detected. In particular, many binding assays use luminescence energy transfer measurements to detect the formation or breakdown of a bound complex between appropriately labelled species. The most common technique for such assays is based on a system where one component of the complex (the xe2x80x98donorxe2x80x99) is labelled with a photoluminescent label and a second component (the xe2x80x98acceptorxe2x80x99) is labelled with a species (which might or might not be luminescent) having an absorption spectrum which overlaps with the luminescence emission of the first species. When the labelled species approach sufficiently closely a radiationless resonant transfer of energy takes place such that the luminescence of the excited donor species is wholly or partially quenched while the acceptor species is excited. Radiationless transfer of energy of this type is only efficient when the labelled species approach each other within a few nanometres and the most common example, energy transfer between a fluorescent donor and an acceptor, has an efficiency which decreases as the sixth power of the separation between donor and acceptor. Proximity assays based on resonance energy transfer are very well known in the literature and have found commercial applications, Most often the assays are conducted to detect binding between labelled species, either directly (e.g., a hybridisation between oligonucleotides) or mediated by an analyte of interest (e.g. a xe2x80x98sandwichxe2x80x99immunoassay), though other formats are also used. An / enzyme, for example, might be assayed on the basis of its ability to catalyse formation or breakdown of a covalent linkage between the donor and acceptor, leading to a change in the level of energy transfer between them. There are very many individual combinations of labelled donor and acceptor species bearing recognition ligands such as antibodies, lectins, various peptides, proteins and glycoproteins, nucleic acids, biotin, avidin, and the like. These are used in a variety of formats well known in the art, based for example on detection of binding of a labelled species or on competitive processes where one or more labelled species is displaced by an analyte.
Energy transfer assays frequently are conducted using a luminescent acceptor. detecting the sensitised emission consequent on energy transfer from the donor species. In many cases the assays preferably are conducted in a so-called homogeneous format, where the analyte is introduced into the measurement system and the assay is conducted without physical separation of the bound complex. Homogeneous assays are very convenient, and lend themselves well to automation, but there are potential problems with this approach. There might be coloured materials present in the assay medium which can absorb at the wavelengths of either or both of the donor and acceptor species, or which can quench luminescence from either or both of these by radiationless energy transfer. Equally, there might be other quenching species in the assay medium. The assay medium might be luminescent, as is found in serum samples for example where bilirubin fluorescence is common, and such emission might overlap that of donor and/or acceptor species. These problems are minimised if the sample can be diluted sufficiently to reduce effects due to coloured species and other quenchers, and if the luminescence of the donor and acceptor are distinguishable from that of background. The luminescence assay must be sufficiently sensitive that such dilution does not reduce signal-to-noise ratio to an unacceptable degree.
A further problem with energy transfer assay is common to homogeneous formats and to assays where separation steps can be included. In most assays where sensitised luminescence from the acceptor is measured, there is also some probability of direct excitation of the acceptor by the light used to excite the donor species. This means that the acceptor fluorescence due to energy transfer is detected against a background of directly excited emission which reduces the dynamic range of the assay since small changes above background cannot readily be measured. In additions there might be some level of overlap between the long wavelength xe2x80x98tailxe2x80x99 of the emission from the donor species and the spectral region where acceptor emission is detected.
It is a purpose of the present invention to minimise the effects of these potential difficulties, and to provide adequate sensitivity for assay of diluted samples where necessary.
It is important to understand that sensitivity in the context of luminescence detection is rarely limited by the ability to detect a signal. Photon counting methods are easily able to detect single atoms and molecules of fluorescent substances. It is the ability to reject unwanted background signals that sets the limit of sensitivity for most measurements, and this is a particular problem in homogeneous assay formats where many potentially luminescent species might be present in the assay medium. Consequently, sensitivity is determined by the selectivity of detection. The present invention provides a means to increase selectivity of detection in the context of luminescence energy transfer.
The invention describes an improvement in the conduct of a measurement of proximity between luminescent species based on detection of transfer of excitation energy between them wherein
a first photoluminescent species (the xe2x80x98donorxe2x80x99) and a second photoluminescent species (the xe2x80x98acceptorxe2x80x99) are provided and are such that the donor species and the acceptor species have at least some excitation spectral regions which differ and that at least a part of the emission spectrum of the donor overlaps with at least a part of the excitation spectrum of the acceptor
the donor species is excited with a cyclical temporal sequence of wavelength bands, optionally provided as pulses or modulated in intensity, giving rise to a characteristic temporal fluctuation in emission therefrom and
emission in at least one wavelength band characteristic of the acceptor is analysed to detect the presence of the said characteristic fluctuation or a subcomponent thereof and optionally also to detect a fluctuation characteristic of direct excitation of the acceptor.
Further features of the invention are defined in the appended claims.
The invention is useful in assay formats based on radiative or radiationless transfer of excitation energy between a donor and an acceptor species.